Process for regulating the hydrogen ion concentration of fermentation mashes



Patented Mar. 9, 1937 UNITED STATES PATENT OFFICE PROCESS FOR REGULATINGTHE HYDRO- GEN ION CONCENTRATION OF FERM'EN- TATION MASHES Hugh R.Stiles, Terre Hante, Ind., assignor to Commercial Solvents Corporation,Terre Hautc, Ind., a corporation of Maryland I No Drawing.

Application October 2, 1933,

Serial No. 691,889

8 Claims.

ate products and/or end products of an acidic' nature are produced; themetabolic functions 01' the organism are favored by a hydrogen ionconcentration in the mash lower than that secured by the normal activityof the microorganisms themselves. In such cases, it has been customaryto add to the mash an insoluble neutralizing agent such as calciumcarbonatabarium carbonate or the like. With a material of this naturepresent in the mash, the hydrogen ion concentration secured by theaction of the bacteria approaches more closely the desired value. It hasbeen noted in many fermentations of this nature that maximum yields areobtained only when the final hydrogen ion concentration secured by theaction of the bacteria falls within a rather narrow optimum range. Theoptimum value can, in some cases, be obtained simply by the use of anexcess of coarsely ground calcium carbonate. In case of otherfermentations, however, this has been found under certain circumstancesto be insufficient. For example, in copending application Serial No.710,897 by D. A. Legg and H. R. Stiles, filed February 12, 1934, it isshown that in certain circumstances very finely divided calciumcarbonate must be employed in order to'secure optimum yields.

However, there are a number of fermentations which require formaximum-yields a final hydrogen ion concentration even lower than thatsecured by the bacteria when finely divided calcium carbonate is presentin the mash. A typical fermentation of this type is the butyl-acetonic45 fermentation of inverted carbohydrate mashes by means of organisms ofthe type Clostridium inverto-acetobutylicum (described in copendingapplicatlon Serial No. 675,458 by D. A. Legs and H. R. Stiles, filedJune 12, 1933).-

50 The bacteria designated in this application as Clostridium invrto-acetobutylicum comprise any bacteria having the followingcharacteristics:

I. Morphological B. spore-formingclostrldia and Plectridia C.Practically indistinguishable from members of the Clostridium buturicumgroup II. Biochemical A. Carbohydrate fermentation 1.' Inability toproduce appreciable yields of butyl alcohol and acetone from starch asthe only source of carbohydrate 2. Inability to produce appreciableyields of butyl alcohol and acetone from sucrose as the only source ofcarbohydrate 3. Inability to consistently produce yields greater than20% on the weight of thesugar from uninverted molasses '4. Ability toproduce high yields of butyl alcohol and acetone from glucose orinverted molasses B. Nitrogen metabolism 1. Ability to produce highyields of butyl alcohol and acetone in sugar media containing ammonia asthe principal source of nitrogen 2. Ability to utilize degraded protein(including ammonia) as the sole nitrogen source 3. Inability to utilize'undegraded protein as sole source of nitrogen 4. Inability to liquefygelatin or to produce more than very slight proteolysis of milk n C.Oxygen requirements 1. Anaerobic D. Temperature range for solventproduction 1. From 25 C. to 36 (3., preferably 29 C.

to 31 C. E. Hydrogen ion concentration for solvent production g 1. FinalpH of 5.0-6.5, preferably 5.7-6.1 The optimum hydrogen ion concentrationfor this fermentation is somewhat more critical than in the usual typesof fermentation having acidic intermediate products, and the yield willbe found "to drop markedly if the final hydrogen ion concentrationsecured by the action of the bacteria varies only slightly from theoptimum. ,Numerous attempts have been made to secure a better regulationof the hydrogen ion concentration in fermentations of this type, butuntil the present time all of these attempts have failed. For example,continuous or intermittent introduction of neutralizing agents durin thfermentation has been unsatisfactory, owing to the mechanical dimcultiesinvolved. A large excess of insoluble neutralizing agent was found tohave no beneficial effect and in some cases actually a detrimentaleiiect. It was thought that the addition of a soluble alkaline materialto the mash prior to inoculation might have a beneficial effect, butsuch materials as sodium carbonate, sodium silicate, trisodiumphosphate, and borax were found to be ineffective.

However, I have now found that, although most soluble alkaline materialsare unsuitable for this purpose, ammonia or a basic ammonium salt, whenadded to the fermentation mash in addition to the insoluble neutralizingagent, gives rise to markedly improved results. The final hydrogen ionconcentration in the mash, secured by the action of the bacteria whenboth an insoluble neutralizing agent and a basic ammonium compound arepresent, is considerably lower than that previously obtainable with anydegree of consistency, resulting in substantially higher yields thanthose previously obtainable. The theory according to which my inventionoperates is not definitely understood, but at least it is clear that theimproved results are not due to the nutrient value of the added ammonia.Improved yields are obtained, according to my invention, in mashes inwhich a sufficient amount of ammonia for nutrient purposes is alreadypresent in the form of a neutral salt.

My invention may probably best be illustrated by the following specificexample: A molasses mash containing 323 grams of Louisiana molasses(165.2 grams of sugar) in 795 c. c. of water was inverted by heatingwith 3.85 c. c. of 95% sulphuric acid for 40 minutes at 20 lbs.pressure. At the conclusion of the inversion the acid was partiallyneutralized by means of 6.45 c. c. of ammonium hydroxide containing21.4% NHs, and 18 grams of precipitated calcium carbonate were thenadded. This calcium carbonate served to neutralize the remainingsulphuric acid and to supply an excess for neutralizing purposes duringthe fermentation. The mash was then diluted to a volume of 3100 c. c.and sterilized for 30 minutes at 20 lbs. pressure. Duplicatefermentations of the above ,quantity of mash in 4 liter flasks werecarried out in each case. If ammonia (in the form of ammonium hydroxide)was utilized as the additional neutralizing agent, this was addedaseptically after sterilization. If ammonium salts were used, these wereadded to the mash prior to sterilization. All of the flasks wereinoculated with 4% of an active culture of the type C'Zostridiuminverto-acetobutylicum and incubated at 30 C. The results of thefermentations are reported in the table below.

Table Solvent ratio Additional Solvent N neutralizing FlIlBl yield agentgrams pH percent Butyl A mm Ethyl per liter on alcohol went alcoholsugar percent pe percent None 5. 65 25. 9 69. 9 27. 7 2. 4 (1%H;)2HP045. 99 30. 68. 6 29. 1 2. 3

.6 g. (biHOaHPOL. 6.00 30. 5 68. 6 29. 2 2. 2

. g. (N]%(|])2HPOA 6. 05 30. 1 68. 1 29. 5 2. 4

2. g. (NHl):COa-.-- 6. 03 29. 2 67. 4 30. 5 2.1

1. g. (NH&)2CO:..-- 6.15 28.3 66. 4 30. 6 3.1

2.00 g. NHIa i 5. 75 29. 2 67. 4 30. 0 2. 6

0. g. NH: 5. 76 30. 2 67. 7 30. 1 2. 2

0.21 g ,m H: 5. 83 30. 4 67. 1 30. 8 2. 1 0.28 g.

It may seen from the above results that,

when practicing my invention, the final pH in the mash falls within therange 5.7-6.1, the optimum range for organisms of this group, and thatthe yields obtained in these fermentations were markedly improved. Itshould be noted that in any particular case there is an optimumconcentration of the ammonium compound employed. Amounts in excess ofthis value, while further decreasing the final hydrogen concentration,apparently tend to decrease the yields somewhat. The amount of theammonium compound to be employed in any particular mash may readily bedetermined by one skilled in the art by simple preliminary experiments.This amount may be found to vary in different cases, depending upon theconcentration of similar materials in the molasses or other raw materialemployed, but in general it will be found to fall within the range0.1-0.3 gram per liter of NH; or its equivalent in the form of a basicammonium salt. For example, 0.1-0.3 gram of NH3,1I1 the form of NH4OH,per liter or 0.3-1.5 grams ,of (NH4) 2HPO4 per liter will usually befound to be satisfactory. In any case, an amount of the basic ammoniumcompound substantially smaller than the amount of insoluble neutralizingagent should be employed.

It is to be understood, of course, that my invention is not to be.limited to the particular example given above. This example wasillustrative only, and my invention is applicable to various other typesof mashes and fermentation processes. For example, my invention may beapplied to the fermentation of any mash prepared in the usual mannerwhich contains a fermentable carbohydrate and suitable nutrients for thebacteria to be employed. Likewise, my invention may be carried out inconjunction with the use of any substantially water-insoluble, non-toxicneutralizing agent. Although the particular example given above waslimited to the use of bacteria of the type Clostridiuminverto-acetobutylicum, it will be evident to those skilled in the artthat my invention is applicable to a number of other fermentations inwhich acidic intermediate products and/ or end products are produced. Ingeneral, it may be said that my invention is applicable to fermentationsby means of organisms whose metabolic functions are favored by ahydrogen ion concentration lower than that obtaining when an excess ofan insoluble neutralizing agent in the mash constitutes the only meansfor hydrogen ion control.

As used in the appended claims, theterm basic ammonium compound" is tobe taken as including ammonia itself either in the gaseous form or insolution as ammonium hydroxide. By the term substantiallywater-insoluble basic neutralizing agent, as used herein and in theappended claims, is meant an alkaline neutralizing agent sufficientlyinsoluble so that the hydroxyl ion concentration of a mash containing anexcess of such neutralizing agent over that required to neutralize anyinitial acidity in the absence of soluble alkaline materials does notexceed neutrality.

My invention now having been described, what I claim is:

1. In a. process for the fermentation of a carbohydrate mash by means ofbacteria whose metabolic functions are favored by a hydrogen ionconcentration lower than that obtained by the use of an excess of aninsoluble basic neutralizing agent over that required to neutralize anyinitial acidity, the improvement which comprises carrying out thefermentation in the presence of said excess of a substantiallywater-insoluble, nontoxic neutralizing agent and approximately 0.2 gramper liter NH; equivalent of a basic ammonium compound.

2. In a process for the fermentation of a carbohydrate mash by means ofbacteria whose metabolic functions are favored by a hydrogen ionconcentration lower than that obtained by the use of an excess of aninsoluble basic inorganic neutralizing agent over that required toneutraling out the fermentation in the presence of said excess ofprecipitated calcium carbonate and from 0.1 03 gram per liter NH:equivalent of a basic ammonium compound.

4. In a process for the production of normal butyl alcohol, acetone, andethyl alcohol by the fermentation of an inverted carbohydrate mash bymeans of bacteria of the group Clostfidium inoerto-acetobutylicum, theimprovement which comprises carrying out the fermentation in thepresence of an excess of a substantially waterinsoluble, non-toxic basicneutralizing agent over that required to neutralize acidity andapproximately 0.2 gram per liter NHa equivalent of a basic ammoniumcompound.

5. In a process for the production of normal I butyl alcohol, acetone,and ethyl alcohol by the fermentation of an inverted carbohydrate mashby means of bacteria of the group Clostridium inoerto-acetobutylicam,the improvement which comprises carrying out the fermentation in thepresence of an excess of a substantially water-insoluble, non-toxicbasic inorganic neutralizing agent over that required to neutralize anyinitial acidity and from 0.1-0.3 gram per liter NHa equivalent of abasic ammonium compound.

6. In a process forthe production of normal butyl alcohol, acetone, andethyl alcohol by the fermentation of an inverted carbohydrate mash bymeans of bacteria of the group Clostridium.

inverto-acetobutylicum, the improvement which comprises carrying out thefermentation in the presence of an excess of precipitated calciumcarbonate over that required to neutralize any initial acidity and from0.l-0.3 gram per liter NH: equivalent of a basic ammonium compound.

'I. In a process for the production of normal butyl alcohol, acetone,and ethyl alcohol by the fermentation of an inverted carbohydrate mashby means of bacteria of the group Clastrirtium inverto-acetobutylicum,the improvement which comprises regulating the hydrogen ionconcentration of the mash by means of an excess of substantiallywater-insoluble, non-toxic basic inorganic neutralizing agent over thatrequired to neutralize any initial acidity and from 0.l-0.3 gram perliter NH: equivalent of a basic ammonium compound whereby the finalhydrogen ion concentration secured by the action of the bacteria fallswithin the range pH 5.7 and pH 6.1.

8. In a process for the productlonof normal butyl alcohol, acetone, andethyl alcohol by the fermentation of an inverted carbohydrate mash bymeans of bacteria of the group Clostridium inverto-acetobutylicum, theimprovementwhlch comprises regulating the hydrogen ion concentration ofthe mash by means of an excess of precipitated calcium carbonate overthat required to neutralize any initial acidity and from 0.1-0.3 gramperliter NH; equivalent of a basic am- ,monium compound whereby thefinal hydrogen ion concentration secured by the action of the bacteriafalls within the range pH 5.7 and pH 6.1.

HUGH R. 5mm.

